Device and method for large volume transfection

ABSTRACT

Disclosed is a device for applying an electric field to a suspension of cells, comprising at least one chamber which comprises at least one internal space (40) for holding the suspension, the internal space (40) comprising at least two segments (41, 42), wherein each segment (41, 42) comprises at least one electrode (43, 44) and wherein neighboring electrodes (43, 44) are separated from each other by at least one gap (47) which is at least partially filled with an insulating material (46), and wherein the edges of the electrodes (43, 44) facing each other within the internal space (40) are rounded. Rounding the electrodes&#39; edges facing a neighboring electrode results in a significant reduction of field gradients and thus even of the risk of arcing. Also disclosed is a method in which voltage is applied to at least one active electrode (43, 44) while the electrodes (43, 44, 45) or electrode segments next and/or opposite to the active electrode (43, 44) are set to ground potential. Setting neighboring electrodes that surround the active electrode to ground potential results in decreased scattering of the electric field within the internal space so that the electrically active area is locally limited and the field lines are focused near the active electrode and thus control of the process is enhanced.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application of the U.S. application Ser. No.15/308,366, filed Nov. 2, 2016, which is a national stage ofInternational application PCT/EP2015/059152, filed Apr. 28, 2015designating the United States, which are incorporated herein byreference in their entireties, and claims priority to Europeanapplication EP 14166918.4, filed May 2, 2014 and to European applicationEP 14191272.5, filed Oct. 31, 2014.

BACKGROUND OF AND INTRODUCTION TO THE INVENTION

The invention relates to a device for applying an electric field to asuspension of cells, cell derivatives, organelles, sub-cellularparticles and/or vesicles, comprising at least one chamber whichcomprises at least one internal space for holding the suspension, theinternal space comprising at least two segments, wherein each segmentcomprises at least one electrode and wherein neighboring electrodes areseparated from each other by at least one gap which is at leastpartially filled with an insulating material. The invention furtherconcerns a method for applying an electric field to a suspension ofcells, cell derivatives, organelles, sub-cellular particles and/orvesicles, wherein a voltage is applied to electrodes of a chambercomprising at least one internal space for holding the suspension, theinternal space comprising at least two segments, wherein each segmentcomprises at least one electrode.

The introduction of biologically active molecules, for example DNA, RNAor proteins, into living cells, cell derivatives, organelles,sub-cellular particles and/or vesicles may, e.g., serves to examine thebiological functions of these molecules and is, moreover, an essentialprecondition for the success of the therapeutic use of these molecules,e.g., in gene therapy. A preferred method for introducing externalmolecules into the cells is called electroporation, which unlikechemical methods limits undesirable changes in the structure andfunction of the target cell. In electroporation the external moleculesare introduced into the cells from an aqueous solution, preferably abuffer solution specifically adapted to the cells, or a cell culturemedium, via a short current flow, i.e., e.g., the pulse of a dischargingcapacitor which renders the cell membrane transiently permeable to theexternal molecules. The temporary “pores” that are formed in the cellmembrane allow the biologically active molecules to first reach thecytoplasm in which they may already perform their function or exert anytherapeutic action to be examined, and then, under certain conditions,to also reach the cell nucleus as it is required, e.g., in gene therapyapplications.

Due to a short application of a strong electrical field, i.e. a shortpulse with a high current density, cells, cell derivatives, organelles,sub-cellular particles and/or vesicles may also be fused. In thisso-called electrofusion the cells are, e.g., initially brought intoclose membrane contact by an inhomogeneous electrical alternating field.The subsequent application of an electrical field pulse leads tointeraction between membrane parts, which ultimately results in fusion.Devices comparable to those used for electroporation may be used forelectrofusion as well.

Smaller volumes of suspension of cells, cell derivatives, organelles,sub-cellular particles and/or vesicles are generally treated in a batchprocess in relatively simple vessels. The solution or cell suspension,respectively, is frequently located in a cuvette, i.e. a narrow vesselopen at the top, which in the vicinity of the bottom has two opposing,parallel electrodes in the lateral walls which serve to apply theelectrical voltage. However, such vessels are unsuitable for treatinglarger volumes as the reaction space available for the electricaltreatment is limited by the limited maximal distance between theelectrodes. Thus, flow-through processes in which the cell or vesiclesuspension is continuously or discontinuously fed through the reactionspace between the electrodes are often used for the electroporation orelectrofusion of larger volumes.

WO 2011/161092 A1 discloses an electrode assembly for applying anelectric field to adherent cells growing at the bottom of a container.The electrode assembly is designed to be inserted into the container andcomprises a plurality of electrodes, each having a surface which isarranged opposite the corresponding surface of the next electrode. Thegap between these surfaces is completely filled with an electricallyinsulating material so that the electric field is concentrated in theregion of the cells to be treated such that a voltage pulse, or thecurrent produced thereby, flows through the cells.

US 2007/0128708 A1 discloses a scalable device for electroporatingrelatively large volumes of a fluid medium carrying biological cells orvesicles in a segmented chamber, wherein each segment comprises twoelectrodes. The effective volume of the chamber can be varied by movinga plunger along the longitudinal axis of the chamber. Thus, the volumechosen is directly related to the volume of the sample to beelectroporated. The sample is sucked in and purged out of the chamberthrough a port disposed in the end wall of the chamber. The samplewithin the chamber is processed by sequentially applying voltage pulsesto the electrode pairs of the individual segments of the chamber.

However, it is a drawback of the prior art devices and methods that therisk of arcing is increased, especially if high voltages are applied tosegmented electrodes, and that the electric field lines can spread outinto regions aside the active electrode segment(s).

It is therefore an object of the invention to provide a device and amethod for treating cells, cell derivatives, organelles, sub-cellularparticles and/or vesicles, with segmented electrodes for keepingelectrical currents low, and with which the risk of arcing is reducedand the electric field is confined to the region near the activeelectrode segment(s).

SUMMARY OF THE INVENTION

The object is met by a device for applying an electric field to asuspension of cells, cell derivatives, organelles, sub-cellularparticles and/or vesicles as initially specified, with which the edgesof the electrodes facing each other within the internal space arerounded. If voltage is applied to the electrodes, the risk of arcing issignificantly increased in regions of sharp contour changes (edges) orif inhomogeneity of the electric field occur very close to the electrodesurface of an active segment. Surprisingly, rounding the electrodes'edges facing a neighboring electrode results in a significant reductionof such field gradients and thus even of the risk of arcing. Accordingto the invention, homogenization of the electric field within theinternal space of the chamber and especially near the electrode surfacein the region of the gaps between electrode segments is achieved by theprovision of a smooth shape transition from a first electrode surfacefacing the lumen of the internal space to a second electrode surfaceperpendicular to the first electrode surface, whereby the secondelectrode surface is facing the electrode gap. In particular the smoothshape transition is provided by a curved electrode surface, i.e. from alarger to a smaller fillet radius (e.g. several tangentially linkedcircle segments or a spline).

Moreover, reduction of field gradients and homogenization of theelectric field also results in decreased scattering of the electricfield within the internal space. Accordingly, the rounded edges of theelectrodes facing each other within the internal space of the chamberhave the surprising effect that high field densities are avoided.

According to an exemplary embodiment of the invention the fillet radiusof the rounded edges of the electrodes is maximized. Surprisingly, ithas been found out that reducing the inhomogeneity of the electric fieldby maximizing the fillet radius of the rounded edges results in asignificant decrease of the likelihood of arcing. That is, the largerthe radius of the rounded edges, the lower the risk of arcing.

In another exemplary embodiment of the invention the width of the gapand/or the distance between two neighboring electrodes is minimized. Asthe cells, cell derivatives, organelles, sub-cellular particles and/orvesicles are not sufficiently processed in the internal space around thegap, the gap (i.e. the distance between two neighboring electrodes)should be as small as possible.

Accordingly, the smaller the width of the gap, the higher the efficiencyof processing.

For example, the design of the device according to the invention can beoptimized by determining the optimal ratio of fillet radius and gapwidth. That is, the fillet radius of the rounded edges of the electrodeshas to be maximized while the width of the gap has to be minimized. Theideal design ensures a very low risk of arcing and a very highprocessing efficiency.

In an exemplary embodiment, which is suitable for many applications, thefillet radius of the rounded edges of at least one of the electrodes isin the range of about 0.3-2.0 mm. For example, the radius may be in therange of about 0.3-1.8, 0.3-1.6, 0.3-1.4, 0.3-1.2, 0.3-1.0, 0.5-2.0,0.7-2.0, 0.9-2.0, 1.0-2.0, 0.4-1.9, 0.5-1.8, 0.6-1.7, 0.7-1.6, 0.8-1.5,0.9-1.4, or 1.0-1.2.

In the same or another exemplary embodiment, which is also suitable formany applications, the width of the gap and/or the distance between twoneighboring electrodes is in the range of about 0.5-2.0 mm. For example,the width may be in the range of about 0.5-1.8, 0.5-1.6, 0.5-1.4,0.5-1.2, 0.5-1.0, 0.6-2.0, 0.7-2.0, 0.9-2.0, 1.0-2.0, 0.6-1.9, 0.7-1.8,0.8-1.7, 0.9-1.6, 1.0-1.5, 1.1-1.4, or 1.1-1.3.

In another exemplary embodiment of the invention the surface of theinsulating material facing the internal space miters the surface of atleast one electrode in a right angle. By designing the surface of theinsulating material such that it is arranged perpendicular to theelectrode's surface, the equipotential lines of the electric field meetthe surface of the electrode orthogonally and are not deflected. As aresult, remaining inhomogeneity of the electric field can be avoidedwithin the chamber or at least moved to a region within the insulatingmaterial or away from the electrode surface of the active segment sothat the likelihood of arcing is further reduced. Moreover, the maximumfield density close to the active electrode is decreased.

In the same or another exemplary embodiment the design of the deviceaccording to the invention can be optimized by varying the radii for thecurvature of the electrodes in order to maximize the radius of theelectrode surface facing the lumen of the internal space of the chamberand at the same time minimizing the gap width. That is, in an exemplaryembodiment the radius of the electrode surface facing the lumen of theinternal space can be larger than the radius of the electrode surfacefacing the insulating material of the gap.

In particular, in an exemplary embodiment the radius of the electrodesurface facing the lumen of the internal space is in the range of about1.0-2.0 mm, and the radius of the electrode surface facing theinsulating material of the gap is in the range of 0.3-2.0 mm. As afurther aspect of this embodiment the surface of the insulating materialfacing the internal space miters the surface of at least one electrodein a right angle exactly at or in the vicinity of the position of theradius change of the electrode surface curvature.

The insulating material within the gap between two neighboringelectrodes may, for example, comprise or consist of polycarbonate.

In another exemplary embodiment of the invention at least one of theelectrodes is larger than the other(s). For example, the largerelectrode may be a counter or ground electrode that is arranged oppositeto the smaller electrodes. In this embodiment the smaller electrodes canbe either active electrodes that are set to high voltage or electrodesthat are set to ground potential.

In an exemplary embodiment, which is suitable for many applications, atleast one electrode has a width in the range of 5-20 mm and at least oneelectrode has a width in the range of 20-80 mm.

In another exemplary embodiment of the invention the gap is arrangedsuch that a part of at least one electrode is disposed opposite to saidgap. Since in this advantageous arrangement each gap is not arrangedopposite to another gap but instead opposite to an electrode, theregions within the internal space of the chamber which are not exposedto an electric field sufficient for efficient processing are minimizedor even eliminated. As a result, the overall processing efficiency iseffectively increased by this measure.

In yet another exemplary embodiment of the invention each segment isprovided with at least one first electrode and at least one secondelectrode, wherein the second electrode is a common electrode of atleast two segments. Such configuration facilitates construction andassembly of the device according to the invention and further avoidscomplicated wiring.

For example, the chamber of the device according to the invention maycomprise corresponding components which can be attached to each other.That is, the device according to the invention can be assembled, e.g.,by attaching two components to each other, wherein each componentcomprises a recess that corresponds to the recess of the othercomponent. If these two components are attached to each other, theiraligned recesses form the internal space of the chamber. In order to becapable of producing an electric field within the internal space, eachrecess can be provided with at least one electrode. At least some of theelectrodes may be segmented. For example, one half of the electrodes (atone side of the symmetry axis) can be segmented while the other half ofthe electrodes (at the other side of the symmetry axis) can be a single,unsegmented electrode which may be used as a counter electrode. In anadvantageous embodiment the two components are identical so thatcost-effective production is ensured. As the identical components arerotationally symmetric, easy assembly by attaching the components toeach other is ensured.

In an exemplary embodiment of the invention at least one segment has avolume in the range of about 10 μl to 500 μl or 20 μl to 400 μl or 30 μlto 300 μl or 50 μl to 200 μl.

In the same or another exemplary embodiment the lumen of the internalspace of the chamber has a volume of at least 500 μl or at least 800 μlor at least 1 ml.

The invention further relates to a method for producing a device forapplying an electric field to a suspension of cells, cell derivatives,organelles, sub-cellular particles and/or vesicles, for example, thedevice according to the invention as described above, wherein at leastone chamber is provided, which comprises at least one internal space forholding the suspension, the internal space comprising at least twosegments and each segment comprising at least one electrode, wherein aninsulating material is at least partially filled into at least one gapwhich separates neighboring electrodes from each other, and wherein theedges of the electrodes facing each other within the internal space aremachined such that they are rounded. Due to this advantageous design,the risk of arcing if voltage is applied to the electrodes issignificantly reduced.

According to an exemplary embodiment of this method the fillet radius ofthe rounded edges of the electrodes is maximized. In another exemplaryembodiment of the method the width of the gap and/or the distancebetween two neighboring electrodes is minimized. For example, the designof the device according to the invention can be optimized by determiningthe optimal ratio of fillet radius and gap width. That is, the filletradius of the rounded edges of the electrodes has to be maximized whilethe width of the gap has to be minimized. The ideal design ensures avery low risk of arcing and a very high processing efficiency.

In another exemplary embodiment of the method the surface of theinsulating material facing the internal space is formed such that itmiters the surface of at least one electrode in a right angle. Byforming the surface of the insulating material such that it is arrangedperpendicular to the electrode's surface, the equipotential lines of theelectric field meet the surface of the electrode orthogonally and arenot deflected. As a result, remaining inhomogeneity of the electricfield can be avoided within the chamber or at least moved to a regionwithin the insulating material and/or away from the active electrodesurface so that the likelihood of arcing is further reduced. Moreover,the maximum field density close to the active electrode is decreased.

In yet another exemplary embodiment of the invention at least one of theelectrodes integrated in the device is larger than the other(s). Forexample, the larger electrode may be used as a counter or groundelectrode that is arranged opposite to the smaller electrodes. In suchembodiment the smaller electrodes can be either used as activeelectrodes that are set to high voltage or as electrodes that are set toground potential. In this embodiment each segment can be provided withat least one first electrode and at least one second electrode, whereinthe second electrode is a common electrode of at least two segments.Such configuration facilitates construction and assembly of the deviceaccording to the invention and further avoids complicated wiring duringproduction of the device.

In yet another exemplary embodiment of the invention the gap is arrangedsuch that a part of at least one electrode is disposed opposite to saidgap. Since in this advantageous arrangement each gap is not arrangedopposite to another gap but instead opposite to an electrode, theregions within the internal space of the chamber which are not exposedto an electric field sufficient for efficient processing are minimizedor even eliminated. As a result, the overall processing efficiency iseffectively increased by this measure.

The object is further met by a method for applying an electric field toa suspension of cells, cell derivatives, organelles, sub-cellularparticles and/or vesicles as initially specified, wherein the voltage isapplied to at least one active electrode while the electrodes orelectrode segments next and/or opposite to the active electrode are setto ground potential. Setting the neighboring electrodes that surroundthe active electrode to ground potential results in decreased scatteringof the electric field within the internal space so that the electricallyactive area is locally limited and the field lines are focused near theactive electrode and thus control of the process is enhanced, especiallyif large volumes are processed in a segmented chamber.

In an exemplary and advantageous embodiment of the invention the voltageis applied to only one active electrode while all other electrodes orelectrode segments in the internal space are set to ground potential.Setting all electrodes in the internal space of the chamber, but for theactive electrode, to ground potential ensures that the field lines arefocused in the internal space near the active electrode and thus only inthe active segment of the chamber and locally faded out towards theneighboring electrodes.

In another exemplary embodiment of the invention the voltage is appliedto at least two electrodes or electrode segments in the internal spacesequentially. It is an advantage of the invention that each segment ofthe internal space of the chamber can be electrically addressedindividually so that controlled generation of electric fields within thechamber can be precisely achieved. For example, in order to avoid arcingand/or undesired heating of the suspension, voltage pulses can beapplied to different segments sequentially. To this end, each segment isprovided with at least one electrode which can be individually addressedso that voltage pulses can be applied to the segments of a chamber insequence.

For example, the segment closest to an outlet port of the chamber isprocessed as first segment followed by the neighboring segment until thelast segment in this sequence, the segment most distant to the outletport, is being processed. That is, the voltage is at first applied tothe segment closest to an outlet port of the chamber, followed by theneighboring segment until the voltage is applied to the last segment inthis sequence, the segment most distant to the outlet port. In thisexemplary embodiment of the invention the segment closest to the outletport is processed as first segment followed by the neighboring segmentuntil the last segment in this sequence, the segment most distant to theoutlet, is being processed. This processing sequence makes sure that gasbubbles occurring during the application of a high voltage to the cellsuspension do not push unprocessed samples towards and/or out of theoutlet but processed sample only.

In yet another exemplary embodiment of the invention each segment isprovided with at least one first electrode and at least one secondelectrode, wherein the voltage is applied to the first electrode and thesecond electrode is a common electrode of at least two segments.Accordingly, the number of electrodes in the internal space of thedevice can be significantly reduced so that control of the process isfacilitated.

The term “rounded” as used herein refers to a curved and even surfacewherein the shape transition from a flat region to another flat regionis tangential.

The term “active electrode” as used herein refers to an electrode towhich a voltage is applied but which is not set to ground potential. Forexample, an “active electrode” may be an electrode which is set to highvoltage potential.

The term “electrode segment” as used herein refers to a separate part ofa segmented electrode, i.e. an electrode which is divided into differentparts, wherein the separate parts of the segmented electrode are notelectrically coupled to each other.

The term “segment” as used herein refers to an area of the internalspace of a chamber, which comprises at least one electrode or electrodesegment.

The term “active segment” as used herein refers to a segment of achamber, which comprises at least one active electrode.

The invention is further exemplarily described in detail with referenceto the figures.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an exemplary embodiment of an individual component of adevice according to the invention comprising a rotatable adjusting meansand a curved chamber design.

a) Separating element in a position at a lower terminal point

b) Separating element in an intermediate position

FIG. 2 shows a schematic representation of different positions of theseparating element of the device according to FIG. 1 .

a) Position at a lower terminal point

b) Position at an upper terminal point

c) Intermediate position

d) Parking position

FIG. 3 shows a perspective view of the outer side of the deviceaccording to FIG. 1 .

FIG. 4 shows different views of the base member according to FIG. 3 .

a) Inner side of the base member with electrodes;

b) Outer side of the base member with conductive areas.

FIG. 5 shows a schematic cross-sectional view of an exemplary embodimentof a device according to the invention.

a) Internal space comprising 8 segments;

b) A part of the internal space according to a) comprising 2 segments.

FIG. 6 shows a representation of a simulation of an electric field ifhigh voltage is applied to the embodiment of the device according toFIG. 5 .

FIG. 7 shows a representation of a simulation of an electric field ifhigh voltage is applied to a device having a larger gap and/or distancebetween two neighboring electrodes or electrode segments.

FIG. 8 shows a representation of a simulation of an electric field ifhigh voltage is applied to a device with conventional electrode design.

DESCRIPTION OF VARIOUS AND PREFERRED EMBODIMENTS

FIGS. 1 a and 1 b show an exemplary embodiment of an individualcomponent of a device 1 according to the invention. The device 1comprises a base member 2 having a curved recess 3 which is providedwith four electrodes 4, 5. Three of these electrodes are segmentelectrodes 4 while one electrode is a counter electrode 5. The basemember 2 represents one component of the device 1 which is assembled oftwo components that are attached to each other, wherein at least theinner sides of these components are identical. That is, the base member2 and a second base member (base member 30 shown in FIG. 3 ) having anidentical inner side are attached to each other so that the recess 3 anda corresponding recess of the second base member form a chamber 6 forholding a suspension of cells, cell derivatives, organelles,sub-cellular particles and/or vesicles. In this chamber 6 an electricfield can be applied to the cells, cell derivatives, organelles,sub-cellular particles and/or vesicles, e.g., for transferringbiologically active molecules such as nucleic acids or proteins into thecells, cell derivatives, organelles, sub-cellular particles and/orvesicles. To this end, the electrodes 4, 5 of base member 2 and thecorresponding electrodes of the second base member establish electrodepairs, wherein the segment electrodes 4 of base member 2 and anoppositely arranged counter electrode of the second base memberestablish three electrode pairs while the counter electrode 5 of basemember 2 and three oppositely arranged segment electrodes of the secondbase member also establish three electrode pairs. In this configurationthe counter electrode 5 of base member 2 and the counter electrode ofthe second base member are each common electrodes of three segments sothat the chamber 6 comprises six segments, wherein each segment isprovided with one segment electrode and an area of one common counterelectrode.

Two ports 7, 8 are disposed at one end 9 of the chamber 6 and two ports10, 11 are disposed at the opposite end 12 of the chamber 6. One port ofthe upper ports 7, 8 can be used as inlet port for charging the chamber6 and the other port of ports 7, 8 can be used as outlet port fordischarging the chamber 6. Similarly, one port of the lower ports 10, 11can be used as inlet port for charging the chamber 6 and the other portof ports 10, 11 can be used as outlet port for discharging the chamber6. Accordingly, each end 9, 12 is provided with two ports 7, 8, 10, 11through which the respective compartment of the chamber 6 can be filledwith the suspension and/or through which the suspension can be purgedout of this compartment. This configuration allows for simultaneouscharging and discharging of the chamber 6 so that the time necessary forchanging the suspension and hence the time lag between two subsequentelectrical treatments of the suspension is minimized. Provision of theports 7, 8, 10, 11 at opposite ends 9, 12 of the chamber 6 allows foreasily establishing a push-pull mechanism where the suspension can bemoved between the two ends 9, 12 of the chamber 6 so as tosimultaneously charge one compartment at one end 9 of the chamber 6 anddischarge another compartment at the opposite end 12 of the chamber 6.Accordingly, the device 1 is not a flow-through device but a device thatenables charging and discharging of the chamber 6 at the same time by apush-pull mechanism wherein the liquid always leaves the chamber on thesame side as it entered it.

In order to separate the suspension that has already been treated by theelectric field from the suspension to be treated, a separating element13 is provided. The separating element 13 can be moved within thechamber 6 between two terminal points 14, 15 and divides the chamber 6into two compartments if it is in a position between the two terminalpoints 14, 15 as depicted in FIGS. 1 b and 2 c . In the exemplaryembodiment depicted in FIGS. 1 and 2 the separating element 13 comprisestwo parts 16, 17 which are spaced from each other and flank an innerspace 18 comprising a compressible material. The two spaced parts 16, 17are wiper-like fingers so that the separating element 13 is a sealingmember which provides a liquid barrier and/or gas barrier between thedifferent compartments of the chamber 6 if it is in a position betweenthe terminal points 14, 15 (FIGS. 1 b and 2 c ). To this end, theseparating element 13 can be made of a flexible and/or elastic materialso that is also capable of compensating pressure peaks within thechamber 6. The separating element 13 may further comprise sealing lipsfor optimal clearing of the chamber 6. The compressible material thatfills the inner space 18 may be air or any other gas, or a compressiblefoam or cellular material, so as to provide effective pressurecompensation in the chamber 6. Accordingly, the separating element 13also acts as a kind of cushion that balances pressure variations in thechamber 6.

The separating element 13 is coupled to an adjusting element 19 whichoperates and/or controls the separating element 13. That is, theseparating element 13 can be moved within the chamber 6 by means of theadjusting element 19. The adjusting element 19 is disposed outside thechamber 6 so that each compartment of the chamber 6 is devoid of anyinterfering element that might affect the function of the device 1. Theadjusting element 19 comprises a rotatable body 20 which is operativelycoupled with the spaced parts 16, 17 of the separating element 13. Inthis exemplary embodiment the rotatable body 20 is a rotor-like elementthat moves the separating element 13 such that it can perform arotational movement along the double arrow 21. This embodiment ensuresprecise control and constant movement of the separating element 13within the curved chamber 6. The rotatable body 20 is surrounded by agasket 22 sealing the adjusting element 19 against the chamber 6,wherein the rotatable body 20 is connected to the gasket 22 via spokes23 made of an elastic material.

The device 1 further comprises a sealing inlay 24 which extends alongthe outer side of the chamber 6 opposite to the gasket 22 describedabove and seals the compartments 26 and 27 of the chamber 6 against eachother. The sealing inlay 24 is made of an elastic and compressiblematerial, e.g., silicone foam or a similar inert material, so that itenables pressure compensation within the chamber.

Advantageously, the device 1 includes means for fixing the separatingelement 13 outside the chamber 6, so that the scalable chamber 6 can beeasily transformed into a static chamber 6 having a fixed volume asshown in FIG. 2 d . To this end, the separating element 13 is moved bymeans of the adjusting element 19 to a parking site 25 where it isfixed, so as to provide the entire volume of the chamber 6 forprocessing of the suspension in a batch process.

FIGS. 2 a-d show different positions of the separating element 13 of thedevice 1 according to FIG. 1 . The method according to the invention isa scalable process for electrically treating a suspension of cells, cellderivatives, organelles, sub-cellular particles and/or vesicles. In FIG.2 a ) the separating element 13 is set to a position at the lowerterminal point 15. If the separating element 13 is rotated to a positionat the upper terminal point 14 (FIG. 2 b )), a first aliquot of thesuspension is injected into one of the lower ports 10, 11 and thuscharged into the chamber 6. The first aliquot is then processed in thechamber 6 by applying an electric field to the suspended cells, cellderivatives, organelles, sub-cellular particles and/or vesicles.Subsequently, the processed first aliquot is discharged through one ofthe lower ports 10, 11 by rotating the separating element 13 back to theposition at the lower terminal point 15 and, at the same time, a secondaliquot of the suspension is provided into one of the upper ports 7, 8and thus charged into the chamber 6. The second aliquot is thenprocessed in the chamber 6 by applying an electric field to thesuspended cells, cell derivatives, organelles, sub-cellular particlesand/or vesicles. Subsequently, the processed second aliquot isdischarged through one of the upper ports 7, 8 by rotating theseparating element 13 back to the position at the upper terminal point14 and, at the same time, a third aliquot of the suspension is injectedinto one of the lower ports 10, 11 and thus charged into the chamber 6.The third aliquot is then processed in the chamber 6 by applying anelectric field to the suspended cells, cell derivatives, organelles,sub-cellular particles and/or vesicles. This push-pull mechanism withsimultaneous charging and discharging of the suspension can be repeateduntil the whole suspension is treated.

The separating element 13 separates the chamber 6 in two compartments26, 27 if it is in a position between the terminal points 14, 15 (FIG. 2c )), wherein each compartment 26, 27 of the chamber 6 is designed tohold a suspension and comprises two ports 7, 8 and 10, 11 for chargingor discharging the chamber 6. Each compartment 26, 27 can receive andhold an aliquot of the suspension which is movable in and out of thechamber 6 through at the ports 7, 8 and 10, 11. The compartments 26, 27are each further provided with one port 7, 10 through which therespective compartment 26, 27 can be filled with the suspension and withone port 8, 11 through which the suspension can be purged out of thiscompartment 26, 27. When the separating element 13 is rotated, onecompartment 26, 27 of the chamber 6 is filled with an aliquot of thesample, while another aliquot of the sample is discharged and pushed outfrom the other compartment 26, 27. A container for incoming sample canbe connected to an upper and a lower inlet port 7, 10 and an upper and alower outlet port 8, 11 can be connected to a reservoir for processedsample. As becomes apparent from FIG. 2 , the device 1 does not work inflow through-fashion but in a push-pull manner wherein injected sampleis discharged after processing on the same side where it was charged.The chamber 6 possesses six electrode segments, one of which beingalways covered by the separating element 13 and thus is not usable. Forexample, the chamber 6 can take 834 μl per cycle. Thus, in this case,1668 μl can be processed in a complete cycle.

In an advantageous embodiment of the invention the separating element isadjusted such that it covers exactly one or more segment electrodes sothat the same electrical parameters can be established within each otherelectrode segment.

The static variant of the device 1 does not allow the separating element13 to rotate. Instead the separating element 13 is fixed outside thechamber 6 at the parking site 25, not covering any electrode segment asshown in FIG. 2 d . With this variant all six electrode segments can beused and thus 1000 μl sample can be processed. For example, the samplecan be injected at a lower or upper inlet port 7, 10 of the device 1 andcan be collected at the lower outlet port 11. Repetitive filling is notpossible in this state of the device 1.

FIG. 3 shows a perspective view of the outer side of the device 1according to FIG. 1 . The device 1 comprises a base member 30, the innerside of which (not visible) being identical to the inner side of thebase member 2 according to FIG. 1 . The base member 30 represents afurther component of the device 1 which is assembled of two components(base members 2 and 30) that are attached to each other. At its outerside, the base member 30 is provided with connectors 31 for connectingconduits to the ports 7, 8, 10, 11 of the chamber 6 according to FIGS. 1and 2 . One or more containers for the suspension to be processed andone or more reservoirs for processed suspension can be connected to theconnectors 31 via suitable conduits. The suspension can be charged intoand discharged from the chamber by means of a pumping element, e.g., avacuum pump or a peristaltic pump or the like, which may be connected tothe suspension circuit between the container(s)/reservoir(s) and theconnectors 31. In order to render the device 1 compatible with commonconduits and pumping systems, the connectors 31 can be Luer slip or Luerlock connectors.

The adjusting element 19 of the device 1 may be connected to a powerunit (not shown), e.g., an electric motor, via a worm gear, a spur gear,a bevel gear, a gear rod, a belt drive, a square-bar steel, or similargear mechanisms or power transmission elements (not shown).

The base member 30 further comprises a multitude of conductive areas 32for providing electric connection to the electrodes in the chamber. Theconductive areas 32 may comprise an electrically conductive polymer, inparticular a polymer doped with electrically conductive material or anintrinsically conductive polymer. The conductive areas 32 are designedto provide an electrical connection between the electrodes and at leastone electric contact point 33. In this embodiment the conductive areas32 are holes in the base member 30 which are at least partially filledwith the electrically conductive material. The conductive areas 32 areelectrically coupled with at least one electric contact point 33 via atleast one conductive path, e.g., copper tracks on a layer of the basemember (not shown). The electric contact point can be contacted by atleast one electric contact, so as to provide direct or indirect electricconnection to a power source.

FIGS. 4 a and 4 b show different views of the base member 30 accordingto FIG. 3 . The inner surface 34 of the base member 30 is depicted inFIG. 4 a ). Electrodes 4, 5 are attached to the inner surface 34. Threeof these electrodes 4, 5 are segment electrodes 4 while one of theseelectrodes 4, 5 is a larger counter electrode 5. The electrodes 4, 5 areattached and connected to conductive areas 32 which extend from theinner surface 34 to the outer surface 35 of the base member 30. Forexample, the electrodes 4, 5 and the electrically conductive materialwithin the conductive area 32 are made of the same material, e.g., anelectrically conductive polymer, in particular a polymer doped withelectrically conductive material or an intrinsically conductive polymeras described above. The polymer can be molded over the inner surface 34and the conductive area 32 of the base member 30 and extend throughholes of the conductive area 32 as shown in detail in FIG. 5 a ). Theconductive areas 32 are electrically coupled with at least one electriccontact point 33 via at least one conductive path (not shown). Theelectric contact point 33 can be contacted by at least one electriccontact, so as to provide direct or indirect electric connection to apower source. In an advantageous embodiment of the invention the basemember 30 is a Printed Circuit Board (PCB).

FIG. 5 a shows an exemplary embodiment of a part of an internal space 40of an exemplary device according to the invention. For example, theinternal space 40 may be part of the chamber 6 of the device 1 accordingto FIGS. 1 and 2 . The internal space 40 comprises eight segments 41.1,41.2, 41.3, 41.4, 42.1, 42.2, 42.3, 42.4, each comprising an electrode43.1, 43.2, 43.3, 43.4, 44.1, 44.2, 44.3, 44.4. Two further electrodes45.1 and 45.2 are disposed opposite to the electrodes 43.1, 43.2, 43.3,43.4 and 44.1, 44.2, 44.3, 44.4, respectively. Neighboring electrodesare separated from each other by an insulating material 46 whichsurrounds the electrodes 43.1, 43.2, 43.3, 43.4, 44.1, 44.2, 44.3, 44.4and fills each gap 47.1-47.8 between neighboring electrodes. Theinsulating material 46 may, e.g., consist of or at least comprisepolycarbonate, FR4 board or other insulating materials. Thecharacteristics of the edges of the electrodes 43.2 and 43.3 as well asthe characteristics of gap 47.2 are further described in detail withreference to FIG. 5 b . These characteristics described below may alsoapply to the other electrodes 43.1, 43.4, 44.1, 44.2, 44.3, 44.4 andgaps 47.1, 47.3-47.8.

FIG. 5 b shows a part of the internal space 40 according to FIG. 5 acomprising two segments 41.2, 41.3 which each comprise an electrode43.2, 43.3. A further electrode 45.1 is disposed opposite to theelectrodes 43.2, 43.3. The neighboring electrodes 43.2, 43.3 areseparated from each other by an insulating material 46 which surroundsthe electrodes 43.2, 43.3 and fills the gap 47.2 between the neighboringelectrodes 43.2, 43.3. In order to avoid undesired arcing, the edges 48,49 of the electrodes 43.2, 43.3 facing each other within the internalspace 40 are rounded. The rounded edges 48, 49 ensure a significantreduction of disturbing gradients in the electric field. Gradients inthe electric field create unnecessary high local field densities andthus increase the undesired risk of arcing. Moreover, homogenization ofthe electric field within the internal space 40 and especially adjacentto the surface of electrodes 43.2, 43.3 can be achieved by providing asmooth shape transition from a flat electrode surface to a curvedelectrode surface, i.e. from a larger to a smaller fillet radius. Suchelectrode design further results in decreased scattering of the electricfield within the internal space 40 so that the electric field lines arefocused near the electrodes 43.2, 43.3.

The design of the device according to the invention may be optimized bydetermining the optimal ratio of the radius of each rounded edge 48, 49and the width of the gap 47.2. This optimization is accomplished bymaximizing the fillet radius of the rounded edges 48, 49 of theelectrodes 43.2, 43.3 and at the same time keeping the width of the gap47.2 as small as possible. The ideal design ensures a very low risk ofarcing and a very high processing efficiency. For example, the filletradius of the rounded edges 48, 49 of at least one of the electrodes43.2, 43.3 could be in the range of about 0.3-2.0 mm, while the width ofthe gap 47.2, i.e. the distance between the neighboring electrodes 43.2,43.3, can be in the range of about 0.5-2.0 mm.

The surface 50 of the insulating material 46 facing the internal space40 may be formed and aligned such that it miters the surface of each ofthe electrodes 43.2, 43.3 in a right angle. As a result, the surface 50of the insulating material 46 is arranged perpendicular to the surfaceof the electrodes 43.2 and 43.3, respectively. Due to this favorabledesign, the equipotential lines of an electric field within the internalspace 40 meet the surface of the electrodes 43.2, 43.3 orthogonally andare therefore not deflected. Accordingly, potential inhomogeneity of theelectric field can be avoided or at least moved to a region within theinsulating material 46 so that the likelihood of arcing is furtherreduced.

The electrode 45.1 facing the electrodes 43.2, 43.3 is larger than theneighboring electrodes 43.2, 43.3 and arranged opposite to the gap 47.2.That is, no other gap is disposed opposite to the gap 47.2 so that theregion near the gap 47.2 is still exposed to an electric fieldsufficient for efficient processing. The overall processing efficiencyis therefore effectively increased. The electrode 45.1 extends over theentire length of both segments 41.2, 41.3 and thus is a common electrodeof both segments 41.2, 41.3. For example, the larger electrode 45.1 maybe a counter or ground electrode while the smaller electrodes 43.2, 43.3can be either active electrodes that are set to high voltage orelectrodes that are also set to ground potential. Voltage may beapplied, for example, to electrode 43.2 (active electrode) while theneighboring electrode 43.3 and the counter electrode 45.1 are set toground potential. Setting the electrodes 43.3 and 45.1 that surround theactive electrode 43.2 to ground potential results in decreasedscattering of the electric field within the internal space 40 so thatthe field lines are focused near the active electrode 43.2 and thuscontrol of the process is enhanced.

For example, at least one of the electrodes 43.2, 43.3 may have a widthin the range of 5-20 mm while the larger electrode 45 may have a widthin the range of 20-80 mm.

During operation of the device according to the invention, when thesuspension of cells, cell derivatives, organelles, sub-cellularparticles and/or vesicles is processed by generating an electric fieldwithin the internal space 40, the flat (or, alternatively, slightlycurved and/or convex) surfaces 51, 52 of the neighboring electrodes43.2, 43.3 which are in contact with the suspension are the main activesurfaces for the process. The flat surfaces 51, 52 are opposed by thelarger electrode 45.1 which may be used as a counter electrode set toground potential. For example, if high voltage is applied to electrode43.3 and the neighboring electrode 43.2 is set to ground potential, anelectric field with high field strength is generated in segment 41.3between the parallel electrode surfaces, i.e. the flat surface 52 ofelectrode 43.3 and the oppositely arranged flat (or, alternatively,slightly curved and/or convex) surface 53 of electrode 45.1 (FIG. 6 ).Due to the advantageous design of the device according to the invention,the equipotential lines in this area are distributed homogenously sothat the risk of arcing is very low. Basically, the following principleis valid: the more homogenous the distribution of the equipotentiallines, the less risk of arcing. Accordingly, inhomogeneity and fieldgradients have to be avoided in the area of transition from the flatsurface 52 to the rounded surface 49 of electrode 43.3. To this end,according to the invention a smooth and constant shape transition isensured by the provision of a first rounding having a first, largerfillet radius and a second rounding having a second, smaller filletradius. The second fillet radius moves the surface of electrode 43.3away from the opposing electrode 45.1 so as to locally reduce fieldstrength. The rounded edge 49 of electrode 43.3 and the design of thesurface 50 of the insulating material 46 as described above result in asignificant reduction of the risk of arcing. Moreover, the electricfield is focused in segment 41.3 between the flat surface 52 ofelectrode 43.3 and the oppositely arranged flat surface 53 of electrode45.1. The same applies to the neighboring electrode 43.2 if high voltageis applied to electrode 43.2 and electrode 43.3 is set to groundpotential during a subsequent voltage pulse.

As becomes apparent from FIG. 6 , the region near the gap 47.2 is stillexposed to an electric field sufficient for efficient processing. As thevolume of the suspension is processed twice when a subsequent voltagepulse is applied to electrode 43.2, medium field strength within thearea between the gap 47.2 and the opposing electrode 45.1 is desired.The width of the gap 47.2, i.e. the distance between the neighboringelectrodes 43.2, 43.3, is therefore optimized.

If the width of the gap gets too large, cells, cell derivatives,organelles, sub-cellular particles and/or vesicles in the middle of theinsulating gap area are exposed to a field strength lower than half ofthe maximum field strength (e.g. gap 54 between electrodes 55, 56depicted in FIG. 7 ). Thus, material processed twice in this area is notideally processed.

The ideal design of the device according to the invention moves possible“hot spots” with very high field gradients away from the electrodesurface/corners. With conventional electrode and gap design (i.e.straight, rectangular electrodes 57, 58 as depicted in FIG. 8 ) highfield gradients close to the electrodes correlate with a low arcingthreshold and thus a much higher likelihood of arcing events.

What we claim is:
 1. A method for applying an electric field to asuspension of cells, cell derivatives, organelles, sub-cellularparticles and/or vesicles comprising: applying a voltage to electrodesof a chamber comprising at least one internal space holding biologicallyactive molecules and the suspension of cells, cell derivatives,organelles, sub-cellular particles and/or vesicles, the internal spacecomprising at least three segments and at least one counter electrode,wherein each segment comprises at least one segment electrode, whereinthe voltage is applied to one of the segment electrodes which is active(active electrode) while all other electrodes in the internal space areset to ground potential, wherein the biologically active molecules areintroduced into the living cells, cell derivatives, organelles,sub-cellular particles and/or vesicles when the electrical field isapplied.
 2. The method according to claim 1, wherein the voltage isapplied sequentially to at least two segment electrodes or segments inthe internal space.
 3. The method according to claim 1, wherein thesegment closest to an outlet port of the chamber is processed as a firstsegment in a sequence of the at least three segments followed by thesegment neighboring the first segment until the last segment in thesequence, the segment most distant to the outlet port, is beingprocessed.
 4. The method according to claim 1, wherein each segment isprovided with the segment electrode as an at least one first electrodeand with at least one second electrode, wherein the voltage is appliedto the first electrode and the second electrode is a common electrode ofthe at least two segments.
 5. The method according to claim 2, whereinthe segment closest to an outlet port of the chamber is processed as afirst segment in a sequence of the at least three segments followed bythe segment neighboring the first segment until the last segment in thesequence, the segment most distant to the outlet port, is beingprocessed.
 6. The method according to claim 2, wherein each segment isprovided with the segment electrode as an at least one first electrodeand with at least one second electrode, wherein the voltage is appliedto the first electrode and the second electrode is a common electrode ofthe at least two segments.
 7. The method according to claim 3, whereineach segment is provided with the segment electrode as an at least onefirst electrode and with at least one second electrode, wherein thevoltage is applied to the first electrode and the second electrode is acommon electrode of the at least two segments.
 8. The method accordingto claim 1, wherein the suspension held in the at least one internalspace contains said cells, cell derivatives and/or organelles.
 9. Themethod according to claim 1, wherein field lines of the electric filedare focused proximate to the active electrode.
 10. A method for applyingan electric field to a suspension of cells, cell derivatives,organelles, sub-cellular particles and/or vesicles comprising: applyinga voltage to electrodes of a chamber comprising at least one internalspace holding biologically active molecules and the suspension of cells,cell derivatives, organelles, sub-cellular particles and/or vesicles,the internal space comprising at least three segments and at least onecounter electrode, wherein each segment comprises at least one segmentelectrode, wherein the voltage is applied to one of the segmentelectrodes which is active (active electrode) while at least twoelectrodes that surround the active electrode, including the counterelectrode and the segment electrodes that are not the active electrode,are set to ground potential, wherein the biologically active moleculesare introduced into the living cells, cell derivatives, organelles,sub-cellular particles and/or vesicles when the electric field isapplied.
 11. The method according to claim 10, wherein all electrodes inthe internal space, but for the active electrode, are set to groundpotential.
 12. A method for electroporation of cells, cell derivatives,organelles, sub-cellular particles and/or vesicles comprising: applyinga voltage to electrodes of a chamber comprising at least one internalspace holding biologically active molecules and a suspension of thecells, cell derivatives, organelles, sub-cellular particles and/orvesicles, the internal space comprising at least three segments and atleast one counter electrode, wherein each segment comprises at least onesegment electrode, wherein the voltage is applied to one of the segmentelectrodes which is active (active electrode) while at least twoelectrodes that surround the active electrode, including the counterelectrode and the segment electrodes that are not the active electrode,are set to ground potential, wherein the biologically active moleculesare introduced into the living cells, cell derivatives, organelles,sub-cellular particles and/or vesicles when the electric field isapplied.
 13. The method according to claim 12, wherein all electrodes inthe internal space, but for the active electrode, are set to groundpotential.